The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
A variety of alternatives for serological methods are known in the art. The review on Immunoassays for diagnosing HIV by Chappel et al. [Chappel R J, Wilson K M, Dax E M. Immunoassays for the diagnosis of HIV: meeting future needs by enhancing the quality of testing. Future Microbiology 2009, 4, 963-982] illustrates the known techniques rather comprehensively.
Classical techniques like enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA), require extensive washing steps but have a rather high sensitivity and specificity. The downside of such tests is that they require several washing and incubation steps, and thus need quite a lot of hands-on time to perform. Rapid tests, on the other hand, in general, require less time and can be separation free. However, the downside of such tests is that typically rather low sensitivity and specificity can be obtained.
Tian & Heyduk 2009 [Anal Chem. 2009, 81, 5218-5225] have disclosed a homogenous immunosensor design which utilizes bivalent nature of an antibody.
According to Tian & Heudyk 2009 the immunosensor design may find applications in antibody detection, e.g. in diagnosis of autoimmune or infectious disease. The disclosed design employs antigens of antibodies conjugated using flexible linkers with short complementary oligonucleotides each containing a fluorochrome that can form a Foster (or fluorescence) resonance energy transfer (FRET) donor-acceptor pair.
Liu et al. [Anal. Chem. 2008, 80, 7735-7741] proposed homogeneous immunoassays based on two-photon excitation FRET (TPE-FRET). They demonstrated that anti-BSA antibody could be determined using bovine serum albumin (BSA) separately labelled with a donor and acceptor. In vivo or intracellular analysis applications were suggested.
Our previous work [PLOS ONE 2013, 8(5), e62739] described an assay based on measuring TR-FRET between donor-labelled antigen and acceptor-labelled antigen bridged by a specific IgG molecule. The assay disclosed relies on labelling the antigen of interest, in two different reactions, with both acceptor and donor fluorochrome. Since the two labelling reactions are commonly done utilizing different methods for activation, the labelling of antigen with other fluorophores may result in “masking” of the antigens epitopes thus preventing the recognition of the antigen by antibodies.